Monosaccharides that carry an α-linked phosphate group are key intermediates in the Leloir pathway for the synthesis of glycosidic linkages. Indeed, they can be converted into nucleotide sugars that are donor substrates for glycosyltransferases. In vivo, galactose-1-phosphate is converted into UDP-galactose by galactose-1-phosphate uridyl transferase (GALT). Absence of this enzyme results in the accumulation of toxic levels of galactose in the blood, a genetic disorder known as galactosemia (Fridovich-Keil, 2006). UDP-galactose, in turn, is the substrate for galactosyltransferases that are involved in the synthesis of a wide variety of important carbohydrate epitopes in glycoproteins and glycolipids (Varki, 1993).
Glycosyl phosphates have traditionally been synthesized by means of conventional chemical catalysis. Starting in 1937, several procedures using different catalysts and different glycosyl or phosphate donors have been described in the literature (Cori et al., 1937; MacDonald, 1961; Inage et al., 1982; Schmidt et al., 1982; Sim et al., 1993). More information can be found in patent EP0553297: “The preparation of glycosyl phosphate triesters.” Chemical phosphorylation of carbohydrates typically consists of multistep reaction schemes, resulting in a low overall yield, and is not very successful in achieving anomeric selectivity. The development of an enzymatic phosphorylation technology is consequently highly desirable, and has the additional benefit of reducing the amount of waste that is generated in the process (green chemistry).
Enzymes that phosphorylate monosaccharides belong to the class of kinases (phosphotransferases), which require ATP as phosphate donor. Most of these sugar kinases phosphorylate their substrate at the C6 position and not at the anomeric center. The only known exceptions are galactokinase (EC 2.7.1.6) that produces α-D-galactose-1-phosphate, and fucokinase (EC 2.7.1.52) that produces β-L-fucose-1-phosphate. Interestingly, the specificity of galactokinase has been broadened by means of directed evolution to include D-talose and L-glucose as substrates (Hoffmeister et al., 2003), and a patent describing the use of such galactokinase variants has been published: “Sugar kinases with expanded substrate specificity and their use” (WO2005056786). Although a kinase is available for the production of α-D-galactose-1-phosphate, the need for the unstable and expensive ATP as a phosphate donor is a serious drawback for industrial applications.
In spite of their name, glycoside phosphorylases do not actually phosphorylate their substrate but, instead, catalyze the phosphorolysis of di- and polysaccharides to produce phosphorylated monosaccharides. These enzymes are highly attractive as biocatalysts because they only require anorganic phosphate as donor, and have long been used for the production of α-D-glucose-1-phosphate from maltodextrin (Griessler et al., 1996) or sucrose (Goedl et al., 2007). More information can be found in U.S. Pat. No. 6,764,841: “Production process of glucose-1-phosphate.” Unfortunately, the specificity of carbohydrate phosphorylases is very limited and only one enzyme is known to produce α-D-galactose-1-phosphate in Nature, i.e., the lacto-N-biose phosphorylase found in Bifidobacteria (Kitaoka et al., 2005). Lacto-N-biose I or β-D-galactosyl-(1,3)-N-acetyl-D-glucosamine is a structural component of oligosaccharides present only in human milk and is not easy to obtain in large quantities (Nishimoto and Kitaoka, 2007).
Japanese Patent No. 9224691 discloses the production of sugar phosphate, useful as food material, by reacting chitobiose or lactose with phosphoric acid in the presence of cellobiose phosphorylase from Cellvibrio gilvus. Starting from lactose, α-D-galactose-1-phosphate is obtained. However, this is only a side activity of the cellobiose phosphorylase, and both long reaction times (48 hours) and the rather low yield make this enzyme, although scientifically interesting, unsuitable for an industrial use.